CD4+Foxp3+ regulatory T cells (Tregs) play a pivotal role in the control of immune tolerance to self-antigens, allergens, and commensals as well as immune responses to pathogens and tumors. Our recent studies have revealed that Treg function is dependent on the transcription factor forkhead box O1 (Foxo1) mediated in part by Foxo1 suppression of the proinflammatory cytokine IFN-? expression. In the first part of the project, we will explore the mechanisms of Foxo1-induced IFN-? repression in Tregs. Genome-wide analysis of Foxo1 binding sites showed that Foxo1 is recruited to the regulatory elements of Ifng and Irf1, which encodes a transcription factor that promotes IFN-? production in T cells. Foxo1 DNA binding and reporter gene assays will be performed to determine whether Foxo1 inhibits the enhancer and promoter activities of Ifng and Irf1. Proteomics studies of Foxo1-associated proteins demonstrated physical interactions between Foxo1 and the transcription factor Runx3. The precise protein domains that mediate Foxo1 interaction with Runx3 will be mapped, and their role in regulating Runx3-induced IFN-? expression will be studied. Furthermore, the in vivo functions of IRF1, IRF1-induced IL-12R?1, and Runx3 in the control of IFN-? expression and the suppressive activities of Foxo1-deficient Tregs will be determined. In the second part of the project, we will use a Toxoplasma gondii infection model to investigate whether Treg acquisition of IFN-? expression and the associated Treg functional defects are caused by the loss of Foxo1 activities. Completion of these studies will generate mechanistic insights to the novel Foxo1-dependent genetic program that controls Treg function in the immunological steady state and during infection.